GnRH analogs

ABSTRACT

Peptides which include unnatural amino acids and which either promote or inhibit the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. Administration of an effective amount of such peptides that are GnRH antagonists prevents ovulation of female mammalian eggs and/or the release of steroids by the gonads and may be used to treat steroid-dependent tumors. The agonists can be used for control of reproduction processes, to treat precocious puberty, endometriosis, and the like. The peptides are analogs of the decapeptide GnRH wherein there is at least one residue of an unnatural amino acid in the 3-, 5-, 6- and/or 8-positions. Unnatural amino acids having the following formula are incorporated in a preferred group of synthesized peptides: ##STR1## Methods for synthesizing such peptides having the triazole side chains are disclosed wherein one side chain modification (or two simultaneously) is carried out on an amino-substituted phenylalanine residue in a peptide chain which is a part of a peptidoresin.

This invention was made with Government support under grant number HD-13527 and contracts NO1-HD-1-3100 and NO1-HD-0-2906 awarded by the National Institutes of Health. The Government has certain rights in this invention.

This application is a continuation-in-part of U.S. Ser. No. 08/078,965, filed Jun. 17, 1993, and a continuation-in-part of U.S. Ser. No. 08/006,729, filed Jan. 21, 1993, now U.S. Pat. No. 5,296,468 which is a continuation-in-part of U.S. Ser. No. 07/669,695, filed Mar. 14, 1991, now abandoned, which is a is a continuation-in-part of U.S. Ser. No. 07/545,239, filed Jun. 27, 1990, now U.S. Pat. No. 5,169,932, which is a continuation-in-part of U.S. Ser. No. 07/428,827, filed Oct. 30, 1989, now abandoned.

This invention relates generally to peptides having unnatural amino acids and to the preparation of new unnatural amino acids, which may be derived from diamino acids, such as Lys, Orn, Dpr and Dbu. More particularly, it relates to GnRH analogs having such unnatural amino acids which can be prepared either in such fully assembled peptides or for incorporation into such peptides as a part of the usual chain elongation synthesis process.

In one more particular aspect, the present invention relates to peptides which inhibit gonadal function and the release of the steroidal hormones, progesterone and testosterone and also to peptides which promote the release of such steroids, as well as to methods of promoting or preventing ovulation.

BACKGROUND OF THE INVENTION

The pituitary gland is attached by a stalk to the region in the base of the brain known as the hypothalamus. In particular, follicle stimulating hormone (FSH) and luteinizing hormone (LH), sometimes referred to as gonadotropins or gonadotropic hormones, are released by the pituitary gland. These hormones, in combination, regulate the functioning of the gonads to produce testosterone in the testes and progesterone and estrogen in the ovaries, and they also regulate the production and maturation of gametes.

The release of a hormone by the anterior lobe of the pituitary gland usually requires a prior release of another class of hormones produced by the hypothalamus. One of the hypothalamic hormones acts as a factor that triggers the release of the gonadotropic hormones, particularly LH, and this hormone is referred to herein as GnRH although it has also been referred to as LH--RH and as LRF. GnRH was isolated and characterized as a decapeptide some 20 years ago, and it was found that analogs of GnRH having a D-isomer instead of Gly in the 6-position, such as [D-Ala⁶ ]-GnRH (U.S. Pat. No. 4,072,668) having the following formula:

    pGlu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH.sub.2,

have greater binding strength to the receptor and greater biological potency than the native hormone.

Peptides are compounds which contain two or more amino acids in which the carboxyl group of one acid is linked to the amino group of the other acid. The formula for GnRH, as represented above, is in accordance with conventional representation of peptides where the amino terminus appears to the left and the carboxyl terminus to the right. The position of the amino acid residue is identified by numbering the amino acid residues from left to right. In the case of GnRH, the hydroxyl portion of the carboxyl group of glycine at the C-terminus has been replaced with an amino group(NH₂) i.e. the C-terminus is amidated. The abbreviations for the individual amino acid residues above are conventional and are based on the trivial name of the amino acid, e.g. pGlu is pyroglutamic acid, Glu is glutamic acid, His is histidine, Trp is tryptophan, Ser is serine, Tyr is tyrosine, Gly is glycine, Leu is leucine, Nle is norleucine, Orn is ornithine, Arg is arginine, Har is homoarginine, Pro is proline, Sar is sarcosine, Phe is phenylalanine, Ala is alanine, Val is valine, Nva is norvaline, Ile is isoleucine, Thr is threonine, Lys is lysine, Asp is aspartic acid, Asn is asparagine, Gln is glutamine, and Met is methionine. Except for glycine, amino acids of the peptides of the invention are of the L-configuration unless noted otherwise.

There are reasons for desiring to prevent ovulation in female mammalians, and the administration of GnRH analogs that are antagonistic to the normal function of GnRH have been used to suppress or delay ovulation. For this reason, analogs of GnRH which are antagonistic to GnRH are being investigated for their potential use as a contraceptive or for regulating conception periods. GnRH antagonists may also be used for the treatment of precocious puberty and endometriosis. The GnRH superagonists can also be used for these purposes. Such antagonists have also been found useful to regulate the secretion of gonadotropins in male mammals and can be employed to arrest spermatogenesis, e.g. as male contraceptives for treatment of male sex offenders, and for treatment of prostatic hypertrophy. More specifically, GnRH antagonists can be used to treat steroid-dependent tumors, such as prostatic and mammary tumors, and for the control of the timing of ovulation for in vitro fertilization. In the female, they can also be used to treat hirsutism.

On the other hand, GnRH agonists function in the same manner as GnRH in promoting the release of LH and FSH, and agonists which exhibit greater biopotency and/or longer duration of action are considered valuable.

In one aspect, it is desired to provide improved peptides which either are strongly antagonistic to endogenous GnRH and prevent secretion of LH and FSH and the release of steroids by the gonads of mammals or are strong agonists of GnRH. Of particular interest are compounds which are more effective in vivo when administered orally.

SUMMARY OF THE INVENTION

The present invention provides unnatural amino acids that can be prepared de novo or by modifying a previously prepared peptide, but which are preferably prepared as a part of a protected peptide-resin, containing a desired overall sequence which includes one or more amino acid residues having a side chain amino group which is to be modified. The resultant preferred amino acids have a side chain which contains either a modified guanidino group or a guanidino equivalent or a derivative that is obtained by further elucidation of a modified guanidino group, as set forth hereinafter, and most preferably it contains a triazole moiety.

In another particular aspect, the invention provides peptides which inhibit the release of gonadotropins in mammalians, including humans, and it also provides methods for inhibiting the release of steroids by the gonads of male and female mammalians. The invention also provides improved GnRH analogs which are strong agonists of GnRH and can be used to promote the reproduction processes of mammalians. As mentioned above, these GnRH antagonists may be used to inhibit the production of gonadotropins and sex hormones under various circumstances, including precocious puberty, hormone dependent neoplasia, dysmenorrhea, endometriosis and steroid-dependent tumors.

The invention provides unnatural amino acids having the following formula U*: ##STR2## where W is (CH₂)_(n) or ##STR3## n is an integer from 1 to 6 and is preferably 1,2,3 or 4; j is 1,2 or 3; Y═N--CN, N--CONH₉, O, S or CH--NO₂, where R₉ is H, Ac, alkyl (preferably C₁ to C₄), naphthyl, pyridyl, pyrimidyl, pyrazinyl, indolyl, quinolinyl or imidazolyl, which alkyl and cyclic groups are unsubstituted or substituted (preferably by chloro, fluoro, bromo, amino, nitro, alkyl (C₁ to C₄) and alkoxy (C₁ to C₄)); X═NH, O, N₃, M₁ --(CH_(q))_(p) --M₂ or M₁ --(CH₂)_(p') --M₂ (CH₂)_(p") --M₃, where M₁ is NR₁₀, N, O or CHR₃ wherein R₃ is methyl, ethyl, propyl, phenyl, pyridinyl, pyrimidinyl or purinyl; q═1 or 2; p, p' and p" are integers between 0 and 6; R₁₀ is H, methyl, ethyl, propyl, phenyl or substituted phenyl (preferably by Cl, F, NO₂ or NH₂); and M₂ and M₃ ═M₁, COOH, CONH₂, COOR₃ or CN (preferably X is NH or O); R₁ ═H, alkyl (preferably C₁ to C₆ and most preferably C₁ to C₄), modified alkyl (preferably C₁ to C₅, the terminal carbon of which is either substituted with NH₂, OH, Cl, Br or F or is replaced with CF₃ or CF₂ CF₃), alkenyl (preferably C₂ to C₄), such as CH₂ CH═CHR₃, alkynyl (preferably C₂ to C₄), such as CH₂ C.tbd.CR₃, aryl such as benzyl, tolyl, p-amino-benzyl (anilinyl) and pCl-benzyl or a direct bond to X; R₂ ═R₁, OH, NH₂, NHR₁, heterocycle (preferably as illustrated hereinafter) or desR₂, with R₂ being desR₂ when X═N₃. Optionally R₂ and X can be interconnected, or R₁ and R₂ can be connected to each other via a branched or unbranched methylene bridge of type --(CH₂)_(m) -- or --(CH₂)_(m) --M--(CH₂)_(m),--. In such an R₁ -R₂ moiety, m and m' are integers from 1 to 6 and preferably from 1 to 3; and M═NH, O, S or CHR₄, wherein R₄ is lower alkyl or aryl and is preferably methyl, ethyl, propyl, phenyl or pCl-phenyl, with M preferably being O or S. Most preferably, when R₁ and R₂ are interconnected, they form a 5, 6, or 7-member heterocyclic ring with the "N--C--X" portion of the formula U*. If desired to form a cyclic peptide, XR₂ can contain a part of another diamino acid within the same peptide, e.g., the omega amino group of the 5-position residue can be so linked to such an unnatural amino acid residue in the 8-position.

Modification of the specified primary amino function of a given amino acid or peptide is carried out by treatment of either the appropriately protected peptide or the amino acid with an appropriate reagent(s). Peptides or amino acids where Y is N--CN (herein referred to as cyanoguanidines) are prepared by reaction of an amino group with diphenyl cyanocarbonimidate (I): ##STR4## wherein "Q" is used to broadly represent either the major portion of a peptide or an amino acid having a primary amino group (such as the amino acid which is depicted above) as a part of formula U*.

The peptide or amino acid having the N-substituted-N'-cyano-O-phenylisourea moiety (II) can then be either isolated or further functionalized by reaction with a second nucleophile HXR₂ to produce cyanoguanidine-containing peptides or amino acids having the formula (III): ##STR5## For example, ##STR6## For example, where HXR₂ ═H₂ N--CH₂ -pyridine, the result is: ##STR7## This group may also be referred to (IUPAC nomenclature) as N-g-cyano-N-g'-3-methylpyridylguanidino.

Such compounds can be hydrolyzed under acidic conditions to produce compounds which are also biopotent--for example: ##STR8## The hydrolyzed versions, referred to herein as including the N-g'-amido group, can also be synthesized directly by reacting phosgene derivates with moieties having a guanidino function.

If HXR₂ is an amino group of another peptide or protein, one will obtain a peptide-peptide dimer or peptide-protein dimer conjugated via the cyanoguanidine moiety. If HXR₂ is the N-terminal primary amino group or the side chain amino group of another amino acid in the same peptide, one will obtain a cyclic peptide (IV) linked via the cyanoguanidine moiety: ##STR9## wherein Q₁ and Q₂ represent the remainders of two amino acid residues in the same peptide. Cyclization via the cyanoguanidine derivative is preferably effected while a part of the peptidoresin, as opposed to subsequently cyclizing the linear peptide.

A special case arises when --XR₂ contains a second nucleophilic site and X has the general form:

M₁ --(CH_(q))_(p) --M₂ or M₁ --(CH₂)_(p'),--M₂ --(CH₂)_(p") --M₃, where M₁, M₂ and M₃ are individually NH, N, O or CHR₃, with p, p', p" being 0,1,2 or 3 and q being 1 or 2. Examples of such nucleophiles include H₂ NNH₂, CH₃ HNNH₂ , CH₃ HNNHCH₃, H₂ NOH, and H₂ N--CH₂ --CH₂ OH. In this case, the cyanoguanidine moiety that is formed can be converted into the corresponding heterocycle (V) which forms from the initial intermediate by reaction of the omega amino group with the cyano group such as: ##STR10## For example, where --XR₂ ═--HNNH₂, ##STR11## Furthermore, where --XR₂ ═--CH₃ NNHCH₃ ##STR12## which may then undergo hydrolysis, as indicated above.

Where XR₂ contains a carboxylic acid group or the equivalent, particularly a carboxylic ester or carboxylic amide, a heterocyclic moiety, such as a saturated pyrimidine-like moiety (VI), is formed, by reaction of the carboxylic group with the secondary amino group (R₁), when M₁ is N, and similar 6-membered heterocyclic moieties are formed when M₁ is O or S. For example, R₂ may be M₁ --(CH₂)_(p) --M₂ with M₂ ═COOH, COOCH₃ or CONH₂ and p being an integer between 1 and 4. For instance in such a case where an aliphatic carboxylic acid group is present and p═2: ##STR13## If R₂ includes an ortho-substituted aromatic carboxylic acid, e.g. benzoic acid (q═1 and p═6), the corresponding quinazoline-like species (VII) is formed: ##STR14## Such benzoic acid may be further substituted, and such substitutions may in any of the other 4 ring positions, as shown, creating the corresponding substituted quinazoline-like moiety which is considered to be equivalent to the unsubstituted. X' may be H, Cl, Br, F, NHCH₃ or SCH₃, and R₇ and R₈ may be H, CH₃ or CH₂ CH₃.

The molecules wherein X═N₃ and R₂ is desR₂ (i.e. deleted) are useful for photolabeling because of the activity of the --N₃ group and are formed by reacting the moiety (II) with sodium azide (NAN₃).

Peptides wherein Y is O (herein referred to as ureas) or S (referred to as thioureas) are prepared by the well known procedure in which the desired side chain amino group is treated with an appropriate isocyanate or thioisocyanate to obtain such ureas or thioureas. ##STR15##

Peptides or amino acids wherein Y is CH--NO₂ (herein referred to as diaminonitroethylenes) are prepared by conversion of the corresponding urea to a carbodiimide: ##STR16## followed by treatment with nitromethane anion (prepared by the action of sodium hydride on nitromethane in dry DMF) as disclosed generally in F. Meimas, et al., Synthesis, 509-510 (1985): ##STR17##

An alternative synthesis that may be used is as follows: ##STR18##

Generally, in accordance with the present invention, peptides are synthesized which are antagonists or agonists of GnRH. Antagonists strongly inhibit the secretion of gonadotropins by the pituitary gland of mammals, including humans, and/or inhibit the release of steroids by the gonads. Agonists strongly promote such secretion or release. These peptides are analogs of GnRH containing one or more unnatural amino acids of the formula U* in the 3-position, the 5-position, the 6-position and/or the 8-position. When U* is in the 3-and/or 6-position, it is always in the form of a D-isomer; whereas when U* is in the 5- and/or 8-position, it is always in the form of an L-isomer. An antagonist should have a 1-position substitution, such as dehydroPro or β-(1-or 2-naphthyl)-D-alanine (hereinafter β-D-1NAL or β-D-2NAL), a 2-position substitution in the form of a modified D-Phe and a 3-position substitution, preferably in the form of substituted or unsubstituted D-Trp, D-3PAL, β-D-NAL or the residue of a D-isomer amino acid U*. The 5-position may be occupied by (a) Tyr, (b) a halogenated or methylated Phe or Tyr, (c) Arg, (d) Lys in which the side chain amino group is acylated by 3-carboxypyridine (nicotinic acid) or by 2 or 4-carboxypyridine, i.e. Lys(cpd), preferably Lys(3cpd) which is also referred to as Lys(Nic), (e) His or (f) the residue of an L-isomer amino acid U*. Agonists have a 6-position substitution which is the residue of the D-isomer U*, and the antagonists may have a D-isomer U* or such a substituted or acylated D-Lys in the 6-position. Instead of Leu in the 7-position, both may have Nle, NML, Phe, Nva, Met, Tyr, Trp or PAL, of which the Phe or Trp may be substituted. The antagonists may also have an optional substitution in the 8-position, which preferably may be the L-isomer U* or isopropyl Lys, i.e., ILys or Lys(Ipr) wherein the side chain amino group is substituted by isopropyl, and a substitution in the 10-position such as D-Ala. At least one residue of a D-isomer amino acid of the formula U* is most preferably present in each peptide of the invention.

Modified D-Phe in the 2-position provides increased antagonistic activity as a result of the specific modifications present in the benzene ring. Single substitutions for hydrogen in the ring are preferably made in the para- or 4-position, but might be in either the 2- or 3-position also; the substitutions are selected from chloro, fluoro, bromo, methyl, methoxy and nitro, with chloro, fluoro and nitro being preferred. Dichloro substitutions are in the 2,4 or 3,4 positions in the ring. The α-carbon atom may also be methylated, e.g. (C.sup.α Me/4Cl)Phe. The 1-position substituent is preferably modified so that its α-amino group contains an acyl group, such as formyl(For), acetyl(Ac), acrylyl(Acr), vinylacetyl(Vac) or benzoyl(Bz), with acetyl and acrylyl being preferred and with acetyl being most preferred. PAL and D-PAL represent the L- and D-isomers of pyridylalanine where the β-carbon of Ala is linked to the 2-, 3- or 4-position, preferably to the 3-position, on the pyridine ring. When β-D-NAL is present in the 1-position and R₅ is not Arg, a hydrophilic D-amino acid residue, such as 4NH₂ -D-Phe, 4-guanidino-D-Phe, D-His, D-Lys, D-Orn, D-Arg, D-Har(Homoarginine) or D-PAL is preferably present in the 6-position if U* is not present. When dehydroPro is present in the 1-position, D-PAL or a D-isomer of a lipophilic amino acid, such as D-Trp, D-Phe, For-D-Trp, NO₂ -D-Trp, D-Leu, D-Ile, D-Nle, D-Tyr, D-Val, D-Ala, dialkyl Arg, dialkyl Har, D-Ser(OtBu), β-D-NAL or (imBzl)D-His is preferably in the 6-position, if U* is not present.

These GnRH analogs are very soluble at a pH just below physiological pH, i.e. about 4.5 to about 6, and thus can be formulated and administered in concentrated form, greatly facilitating administration at a pH of about 5 to 7.4 which is presently preferred. The agonists are substantially more potent than native GnRH in effecting release of LH and FSH. The antagonists inhibit ovulation of female mammals when administered at low levels at proestrus and are also effective to cause resorption of fertilized eggs if administered shortly after conception. The antagonists are also effective for the contraceptive treatment of male mammals and the treatment of steroid-dependent tumors. Certain of the antagonists are surprisingly long-acting in their suppression of LH levels following administration, and certain have a particularly low side effect in respect of histamine release.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As previously mentioned, the unnatural amino acids (which can be L- or D-isomers) are represented by the formula U*: ##STR19## wherein W, X, Y, R₁ and R₂ are as defined previously, and there is at least one such residue (preferably a D-isomer) in each peptide of the invention.

More specifically, the GnRH antagonists of the present invention are represented by the following Formula (F₁):

G-AA₁ -(A)D-Phe-AA₃ -Ser-AA₅ -AA₆ -AA₇ -AA₈ -Pro-AA₁₀ wherein G is hydrogen or an acyl group having 7 or less carbon atoms; AA₁ is dehydroPro, D-pGlu, (A) D-Phe, (B) D-Trp, Pro, or β-D-NAL; A is H, Cl, F, NO₂, CH₃, OCH₃, C.sup.α Me/4Cl, Cl₂ or Br; B is H, NO₂, NH₂, OCH₃, F, Cl, Br, CH₃, N^(in) For or N^(in) Ac; AA₃ is U*, D-PAL, β-D-NAL or (B) D-Trp; AA₅ is U*, Tyr, (C)Arg, Lys(cpd) , Orn(cpd) , Dbu(cpd) , Dpr(cpd), (A) Phe, (3I)Tyr or His; AA₆ is U*, β-D-NAL, (B)D-Trp, (A')D-Phe, (D)D-Orn, (D)D-Lys, (D) D-Dbu, (D) D-Dpr, D-Har, D-Tyr, (E) D-His, D-PAL, (C) D-Arg or a suitable lipophilic D-isomer; A' is A, NH₂, NHCH₃ or gua; C is H or lower alkyl; D is G, cpd or an aryl group; E is H, imBzl or dinitrophenol; AA₇ is Nle, Leu, NML, (A)Phe, Met, Nva, Tyr, (B)Trp or PAL; AA₈ is U*, (C')Arg, (C')Har or ILys; C' is H or di-lower alkyl; AA₁₀ is D-Ala-NH₂, Gly-NH₂, AzaGly-NH₂ or NH(R); R is lower alkyl, preferably CH₂ CH₃ ; and U* is as defined above. When AA₁ is β-D-NAL and AA₅ is not Arg, then AA₆ is preferably U*, 4-NH₂ -D-Phe, D-Lys, D-Orn, D-Har, D-His, 4-gua-D-Phe, D-PAL or D-Arg.

By dehydroPro is meant 3,4 dehydroproline, C₅ H₇ O₂ N. By β-D-NAL is meant the D-isomer of alanine which is substituted by naphthyl on the β-carbon atom, i.e., also 3-D-NAL. Preferably β-D-2NAL is employed wherein the attachment to naphthalene is at the 2-position on the ring structure; however, β-D-1NAL may also be used. The preferred 1-position residues are β-D-NAL, substituted D-Phe and optionally substituted D-Trp. PAL represents alanine which is substituted by pyridyl on the β-carbon atom; preferably the linkage is to the 3-position on the pyridine ring. When substituted D-Trp is employed, single substitutions for hydrogen are preferably made in either the 5- or 6-position, which are selected from chloro, fluoro, bromo, methyl, amino, methoxy and nitro, with chloro, fluoro and nitro being preferred. Alternatively, the indole nitrogen may be acylated, e.g. with formyl (N^(in) For- or 1For-) or with acetyl. D-3PAL, N^(in) For-D-Trp and 6NO₂ -D-Trp are the preferred residues for the 3-position although D-Trp is also often used. When U* is not in the 5-position, Tyr, Arg or Lys(cpd) preferably is present. By NML is meant N.sup.α CH₃ -L-Leu. By Dbu is meant alpha, gamma diamino butyric acid, and by Dpr is meant α,β diamino propionic acid. By Aph is meant 4NH₂ Phe; aminohomophenylalanine and aminohomohomophenylalanine are considered to be equivalents thereof. Instead of being in the para- or 4-position, the amino substitution may also be in the meta or ortho position. When dehydroPro is present in the 1-position, Tyr or U* is preferably present in the 5-position and a lipophilic residue is in the 6-position. By 4-gua-D-Phe is meant a residue of D-Phe having guanidine substituted in the para-position. By AzaGly-NH₂ is meant NHNHCONH₂. The guanidino group of an Arg residue in the 5- or 6-position may be substituted by lower alkyl, i.e. 1 to 4 carbon atoms, e.g., propyl(Pr). When D-Lys, D-Dbu, D-Dpr or D-Orn is present in the 6-position and it is not a part of an unusual amino acid U*, its side-chain-amino group may be acylated by an acyl group which may be aliphatic, heterocyclic or aromatic, e.g. nicotinic acid, or may be substituted by an aryl group having not more than 1 phenyl ring. When U* is not present in the 6-position, it is preferably D-PAL or D-Lys(cpd). The 7-position residue is preferably Leu, NML, Nle or Phe. If the 8-position residue is not U*, it is preferably ILys.

More specifically the GnRH agonists of the invention are represented by the following Formula (F₂): pGlu-His-Trp-Ser-Tyr-U*-AA₇ -Arg-Pro-AA₁₀, wherein U*, AA₇ and AA₁₀ are as defined hereinbefore. Preferably AA₇ is Leu or NML, AA₁₀ is NHCH₂ CH₃ and U* includes a residue of amino-substituted Phe (or its equivalent, e.g homophenylalanine).

Overall, the invention thus provides GnRH analogs having the Formula (F₃):

G-AA-AA₂ -AA'-Ser-AA₅ -AA₆ -AA₇ -AA₈ -Pro-AA₁₀ wherein AA is pGlu or AA₁ ; AA₂ is His or (A)D-Phe; AA' is Trp or AA₃ ; and all others are as defined hereinbefore.

One preferred subgenus of GnRH antagonists has the following formula:

Ac-AA₁ -(A)D-Phe-AA₃ -Ser-AA₅ -AA₆ -AA₇ -AA₈ -Pro-AA₁₀ wherein AA₁ is (A)D-Phe, (B)D-Trp or β-D-NAL; A is H, 4Cl ,4F, 4NO₂, 4CH₃, 4OCH₃, C.sup.α Me/4Cl, 2,4Cl₂ or 4Br; B is H, 6NO₂, 6NH₂, 6OCH₃, 6F, 6Cl 6Br, 6CH₃, N^(in) For or N^(in) Ac; AA₃ is U*, D-PAL, β-D-NAL or (B)D-Trp; AA₅ is U*, Lys(cpd) or Tyr; AA₆ is U*, β-D-NAL, 4NH₂ D-Phe, (B)D-Trp, D-Lys(cpd), D-PAL or D-Arg; AA₇ is Nle, Leu, NML or Phe; AA₈ is U*, ILys, or Arg; AA₁₀ is D-Ala-NH₂, Gly-NH₂, NHNHCONH₂ or NH(R); R is lower alkyl; and U* is either ##STR20## where W is (CH₂)_(n) or ##STR21## n is an integer from 1 to 4; j is 1,2 or 3; X is NH or O; Y is N--CN or N--CONH₉ where R₉ is H or lower alkyl; R₂ is lower alkyl, cyclohexyl, phenyl, pyridyl, methyl pyridyl or histaminyl; or ##STR22## where W is defined as above and R₁₁ is H or an acyl radical having 1 to 6 carbon atoms; provided, however, that at least one of AA₃, AA₅, AA₆ and AA₈ is U*.

Another preferred subgenus of GnRH antagonists has the following formula:

Ac-AA₁ -(A)D-Phe-U*-Ser-AA₅ -AA6-AA7-AA₈ -Pro-AA₁₀ wherein AA₁ is (A) D-Phe, (B) D-Trp or β-D-NAL; A is H, 4Cl, 4F, 4NO₂, 4CH₃, 4OCH₃, C.sup.α Me/4Cl, 2,4Cl₂ or 4Br; B is H, 6NO₂, 6NH₂, 6OCH₃, 6F, 6Cl, 6Br, 6CH₃, N^(in) For or N^(in) Ac; AA₅ is Lys(cpd) or Tyr; AA₆ is β-D-NAL, 4NH₂ D-Phe, (B) D-Trp, D-Lys (cpd), D-PAL or D-Arg; AA₇ is Nle, Leu, NML or Phe; AA₈ is ILys or Arg; AA₁₀ is D-Ala-NH₂, Gly-NH₂, NHNHCONH₂ or NH(R); R is lower alkyl; and U* is either ##STR23## where W is (CH₂)_(n) or ##STR24## n is an integer from 1 to 4; j is 1,2 or 3 (preferably 1); X is NH or O; Y is N--CN or N--CONH₉ where R₉ is H or lower alkyl (C₁ -C₃), preferably H; R₂ is lower alkyl (C₁ -C₆), cyclohexyl, phenyl, pyridyl, methyl pyridyl or histaminyl; or ##STR25## where W is defined as above and R₁₁ is H or an acyl radical having 1 to 3 carbon atoms, preferably acetyl.

Still another preferred subgenus of GnRH antagonists has the formula:

Ac-β-D-2NAL-(4Cl)D-Phe-U*-Ser-Tyr-D-3PAL-Leu-Lys(isopropyl)-Pro-D-Ala-NH₂, wherein U* is a D-isomer having the formula either ##STR26## where R₂ is lower alkyl.

Yet another preferred subgenus of GnRH antagonists has the formula:

Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-U*-U*-AA₇ -Lys (isopropyl)-Pro-AA₁₀ wherein AA₇ is Leu or N.sup.α CH₃ Leu; AA₁₀ is D-Ala-NH₂, Gly-NH₂, or NHCH₂ CH₃ ; and U* is either ##STR27## where R₂ is lower alkyl, pyridyl or methyl pyridyl;

provided, however, that U* in the 6-position is always a D-isomer.

An additional preferred subgenus of GnRH antagonists has the formula:

Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-U*-U*-AA₇ -Lys (isopropyl)-Pro-AA₁₀ wherein AA₇ is Leu or N.sup.α CH₃ -Leu; AA₁₀ is D-Ala-NH₂, Gly-NH₂ or NHCH₂ CH₃ ; and U* is ##STR28## where W is (CH₂)_(n) or ##STR29## n is an integer from 1 to 6; and R₁₁ is H or an acyl radical having 1 to 6 carbon atoms; provided, however, U* in the 6-position is a D-isomer.

One more preferred subgenus of GnRH antagonists has the formula:

Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA₅ -AA₆ -Leu-Lys (isopropyl)-Pro-D-Ala-NH₂ wherein AA₅ is U* or Lys(carboxypyridine); AA₆ is U* or D-Lys(carboxypyridine); and U* is ##STR30## where W is (CH₂)_(n) and n is 4; provided, however, that either AA₅ is U* or AA₆ is U*, with AA₆ always being a D-isomer.

The peptides of the present invention can be synthesized by classical solution synthesis, but are preferably synthesized by a solid phase technique. A chloromethylated resin or a hydroxymethylated resin may be used; however, a methylbenzhydrylamine(MBHA) resin, a benzhydrylamine (BHA) resin or some other suitable resin known in the art which directly provides a C-terminal amide or substituted amide upon cleavage is preferably employed when such a C-terminus is desired. For example, peptides having a substituted amide at the C-terminus are preferably synthesized using an N-alkylamino methyl resin as taught in U.S. Pat. No. 4,569,967, issued Feb. 11, 1986. Solid phase synthesis is conducted in a manner to stepwise add amino acids in the chain in the manner set forth in detail in the U.S. Pat. No. 4,211,693. Side-chain protecting groups, as are well known in the art, are preferably included as a part of any amino acid which has a particularly reactive side chain and optionally in the case of others, such as Trp, which amino acids are to be coupled in the chain being built upon the resin. Such synthesis provides the fully protected intermediate peptidoresin.

Chemical intermediates made generally in accordance with the invention may be represented by the formula: X¹ -AA-AA₂ (X⁵)-U₃ -Ser(X³)-U₅ -U₆ -AA₇ (X² or X⁷)-U₈ -Pro-X⁸ wherein: U₃ is either U' or AA' (X²); U₅ is either U' or AA₅ (X⁴ or X⁵); U₆ is either U' or AA₆ (X⁴ or X⁵ or X⁶); U₈ is either U' or AA₈ (X⁵ or X⁶); U' is either Lys (X^(a)), Aph (X^(a)), Orn (X^(a)), Dbu (X^(a)) or Dpr (X^(a)); X¹ is an α-amino protecting group of the type known to be useful in the art in the stepwise synthesis of polypeptides and when G in the desired peptide composition is a particular acyl group, that group may be used as the protecting group. Among the classes of α-amino protecting groups covered by X¹ are (1) acyl-type protecting groups, such as formyl(For), trifluoroacetyl, phthalyl, p-toluenesulfonyl(Tos), benzoyl(Bz), benzenesulfonyl, dithiasuccinoyl(Dts) o-nitrophenylsulfenyl(Nps), tritylsulfenyl, o-nitrophenoxyacetyl, acrylyl(Acr), chloroacetyl, acetyl(Ac) and γ-chlorobutyryl; (2) aromatic urethan-type protecting groups, e.g., benzyloxycarbonyl(Z), fluorenylmethyloxycarbonyl(Fmoc) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxy-carbonyl(ClZ), p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl and p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups, such as tertbutyloxycarbonyl(Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl and allyloxycarbonyl; (4) cycloalkyl urethan-type protecting groups, such as cyclopentyloxycarbonyl, adamantyloxycarbonyl and cyclohexyloxycarbonyl; (5) thiourethan-type protecting groups, such as phenylthiocarbonyl; (6) alkyl-type protecting groups, such as allyl(Aly), triphenylmethyl(trityl) and benzyl(Bzl); (7) trialkylsilane groups, such as trimethylsilane. The preferred α-amino protecting group is Boc when X is hydrogen.

X² is hydrogen or a protecting group for the indole nitrogen of Trp, such as Bz, Ac or For. In many syntheses there is no need to protect Trp, and such protection is not used if acylated D-Trp is present elsewhere in the peptide.

X³ is hydrogen or a protecting group for the hydroxyl side chain of Ser or Thr, e.g. Ac, Bz, trityl, DCB or benzyl ether(Bzl) and is preferably Bzl.

X⁴ is hydrogen or a protecting group for the phenolic hydroxyl group of Tyr selected from the group consisting of tetrahydropyranyl, tert-butyl, trityl, benzyl, Z, 2-bromobenzyloxycarbonyl(2BrZ) and 2,6-dichlorobenzyl(DCB). 2BrZ is preferred.

X⁵ is a protecting group for a side chain guanidino group, such as that in Arg or Har, or for the imidazole group of His, such as nitro, Tos, trityl, adamantyloxycarbonyl, Z and 2,4-dinitrophenol(Dnp), or X⁵ may be hydrogen, which means there is no protection on the side chain group atoms. Tos is generally preferred.

X⁶ is a protecting group for an amino side chain group, primary or secondary amino, such as Z or 2ClZ; X^(a) is a subclass of X⁶ comprising such protecting groups that can be removed without removing other side chain protecting groups so as to allow the omega-amino group to thereafter take part in the reactions to build the unnatural amino-acid residue. Preferably a base-labile group, such as Fmoc, methylsulfonylethyloxycarbonyl(Msc) or trifluoroacetyl(Tfa), is used; however, it may also be possible to use a hydrazine-labile group such as phthaloyl, ##STR31## or a thiolabile group such as Nps or Dts.

X⁷ is hydrogen or a protecting group for Met, such as oxygen; Met is generally left unprotected.

X⁸ may be Gly-NH-[resin support], D-Ala-NH- [resin support] or N(A)-[resin support]; X⁸ may also be an amide either of Gly or of D-Ala or a substituted amide attached directly to Pro or NHNHCONH₂.

The criterion for selecting side chain protecting groups for X² -X⁷ is that the protecting group should be stable to the reagent under the reaction conditions selected for removing the α-amino protecting group (preferably Boc) at each step of the synthesis. Protecting groups generally should not be split off under coupling conditions but should be removable upon completion of the synthesis of the desired amino acid sequence under reaction conditions that will not alter the peptide chain.

When the X⁸ group is Gly-NH-[resin support] or D-Ala-NH-[resin support], an amide bond connects Gly or D-Ala to a BHA resin or to a MBHA resin. When the X⁸ group is N(A)-[resin support], a substituted amide bond connects Pro to an N-alkylaminomethyl (NAAM) resin. When X⁸ is AzaGly-NH₂, the peptide is preferably made by classical solution synthesis, as disclosed in U.S. Pat. No. 4,234,571.

When G is acetyl, for example, in the final formula, a reaction is preferably carried out with the peptide on the resin (after deblocking the α-amino group while the side-chain groups remain protected), e.g. by reacting with acetic acid in the presence of diisopropyl or dicyclohexyl carbodiimide (DIC or DCC) or preferably with acetic anhydride or by another suitable reaction as known in the art.

In one aspect, the invention particularly provides a method for making a GnRH antagonist peptide having the formula:

G-AA-AA₂ -AA₃ -Ser-AA₅ -AA₆ -AA₇ -AA₈ -Pro-AA₁₀, wherein at least one of AA₅ and AA₆ is U* and the symbols are as set forth hereinbefore, which method comprises (a) forming an intermediate peptide having the formula:

X¹ -AA-AA₂ (X⁵)-AA₃ (X²)-Ser(X³)-Aph(X⁸)-D-Aph(X⁸)-AA₇ (X² or X⁷)-AA₈ (X⁵ or X⁶)-Pro-X⁸ wherein: X¹ is hydrogen or an α-amino protecting group; X² is hydrogen or a protecting group for an indole nitrogen; X³ is a protecting group for a hydroxyl group of Ser or Thr; X⁴ is hydrogen or a protecting group for a phenolic hydroxyl group of Tyr; X⁵ is either hydrogen or a protecting group for a guanidino or imidazole side chain; X⁶ is a protecting group for a primary amino side chain of which X^(a) is a subgroup that is removable without removing other protecting groups; X⁷ is hydrogen or a protecting group for Met; X⁸ is Gly-NH-[resin support] or D-Ala-NH-[resin support]; (b) removing X^(a) to deprotect side chain primary amino groups of the Aph residues of said intermediate peptide; (c) reacting said deprotected side chain primary amino group to build said residue into one having a triazole moiety; and (d) splitting off any remaining groups X¹ to X⁷ and/or cleaving from resin support included in X⁸.

Purification of the peptide is effected by ion exchange chromatography on a CMC column, followed by partition chromatography using the elution system: n-butanol;0.1N acetic acid (1:1 volume ratio) on a column packed with Sephadex G-25, or by using HPLC, as known in the art and specifically set forth in J. Rivier, et al. J. Chromatography, 288 (1984) 303-328.

The antagonists of the invention are effective at levels of less than 100 micrograms per kilogram of body weight, when administered subcutaneously at about noon on the day of proestrus, to prevent ovulation in female rats. For prolonged suppression of ovulation, it may be necessary to use dosage levels in the range of from about 0.1 to about 2.5 milligrams per kilogram of body weight. These analogs are particularly soluble at physiological pHs and thus can be prepared as relatively concentrated solutions for administration. The antagonists are also effective to arrest spermatogenesis when administered to male mammals on a regular basis and can thus be used as contraceptives. Since these compounds will reduce testosterone levels (an undesired consequence in the normal, sexually active male), it may be reasonable to administer replacement dosages of testosterone along with the GnRH antagonist. These antagonists can also be used to regulate the production of gonadotropins and sex steroids for other purposes as indicated hereinbefore.

In the following formulas, the U* residues are defined in terms of the original amino acid residue having a side chain amino group plus the modification in question which is set forth in the accompanying parentheses. Preferably, the original residue is incorporated in the main peptide chain, for example Lys or D-Lys or Orn, Dbu, Dpr or a D-isomer thereof, and is modified while a part of the peptide chain that is still attached to the resin to form the desired residue of the amino acid U*. However, as indicated hereinbefore, the suitably protected unnatural amino acid U* can be added as a part of the usual chain elongation process.

With respect to modified side chain amino groups of the amino acids Lys, Orn, Dbu and Dpr, the following abbreviations are used:

act═acetyl aminotriazole

bcg═aminobutyl cyanoguanidino

bzcg═aminobenzyl cyanoguanidino

bur═N-g-amido, N-g'-butylguanidino

chcg═aminocyclohexyl cyanoguanidino

ecg═aminoethyl cyanoguanidino

icg═aminoisopropyl cyanoguanidino

hcg═aminohexyl cyanoguanidino

hicg═histaminyl cyanoguanidino (ethylimidazole)

mcg═aminomethyl cyanoguanidino

ncg═aminoethyl(1 or 2)naphthyl cyanoguanidino

mncg═aminomethyl(1 or 2)naphthyl cyanoguanidino

Ocg═O-phenyl cyanoguanidino

pcg═aminopropyl cyanoguanidino

Sbcg═thiobutyl cyanoguanidino

tcg═3-amino 1,2,4 triazole

trcg═indole ethylamino cyanoguanidino(tryptamino cyanoguanidino)

mpcg═aminomethyl pyridyl cyanoguanidino (number indicates position of aminomethyl group on pyridyl ring)

The following Examples illustrate a large number of peptides embodying various features of the invention. All of these peptides include at least one D-isomer amino acid residue.

EXAMPLE I

Peptides as indicated in TABLE I having the formula: Ac-β-D-2NAL-(4Cl)D-phe-D-3PAL-Ser-AA₅ -AA₆ -Leu-AA₈ -Pro-D-Ala-NH₂ are prepared by the solid-phase procedure referred to above.

                  TABLE I                                                          ______________________________________                                                AA.sub.5  AA.sub.6     AA.sub.8                                         ______________________________________                                          1       Lys(icg)    D-Lys(icg)   ILys                                          2       Lys(mcg)    D-Lys(mcg)   "                                             3       Lys(chcg)   D-Lys(chcg)  "                                             4       Lys(tcg)    D-Lys(tcg)   "                                             5       Lys(pcg)    D-Lys(pcg)   "                                             6       Lys(2mpcg)  D-Lys(2mpcg) "                                             7       Lys(3mpcg)  D-Lys(3mpcg) "                                             8       Lys(4mpcg)  D-Lys(4mpcg) "                                             9       Lys(hcg)    D-Lys(hcg)   "                                            10       Lys(ecg)    D-Lys(ecg)   "                                            11       Lys(Ocg)    β-D-2NAL                                                                               "                                            12       Lys(bcg)    "            "                                            13       Lys(Nic)    D-Lys(Nic)   Lys(bcg)                                     14       Lys(bcg)    β-D-2NAL                                                                               "                                            15       Lys(bcg)    D-Lys(bcg)   Arg                                          16       Tyr         β-D-2NAL                                                                               Lys(icg)                                     ______________________________________                                    

For purposes of an example, a representative solid phase synthesis of peptide No. 1 above, which is referred to as [Ac-β-D-2NAL¹, (4C1)D-Phe², D-3PAL³, Lys(icg)⁵, D-Lys(icg)⁶, ILys⁸, D-Ala¹⁰ ]-GnRH is set forth hereinafter. This peptide has the following formula:

Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-Lys(isopropyl cyanoguanidino)-D-Lys(isopropyl cyanoguanidino)-Leu-Lys (isopropyl)-Pro-D-Ala-NH₂.

An MBHA resin is used, and Boc-protected D-Ala is coupled to the resin over a 2-hour period in CH₂ Cl₂ using a 3-fold excess of Boc derivative and DCC as an activating reagent. The D-Ala residue attaches to the MBHA residue by an amide bond.

Following the coupling of each amino acid residue, washing, deblocking and coupling of the next amino acid residue is carried out in accordance with the following schedule using an automated machine and beginning with about 5 grams of resin:

    ______________________________________                                                                      MIX                                               STEP  REAGENTS AND OPERATIONS                                                                               TIMES MIN.                                        ______________________________________                                         1     CH.sub.2 Cl.sub.2 wash-80 ml. (2 times)                                                               3                                                 2     Methanol(MeOH) wash-30 ml. (2 times)                                                                  3                                                 3     CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                                                               3                                                 4     50 percent TFA plus 5 percent 1,2-                                                                    10                                                      ethanedithiol in CH.sub.2 Cl.sub.2 -70 ml. (2 times)                     5     Isopropyl alcohol + 1% ethanedithiol                                                                  3                                                       wash-80 ml. (2 times)                                                    6     TEA 12.5 percent in CH.sub.2 Cl.sub.2 -70 ml.                                                         5                                                       (2 times)                                                                7     MeOH wash-40 ml. (2 times)                                                                            2                                                 8     CH.sub.2 Cl.sub.2 wash-80 ml. (3 times)                                                               3                                                 9     Boc-amino acid (10 mmoles) in 30 ml.                                                                   30-300                                                 of either dimethylformamide(DMF) or                                            CH.sub.2 Cl.sub.2, depending upon the solubility                               of the particular protected amino acid,                                        (1 time) plus DIC or DCC (10 mmoles)                                           in CH.sub.2 Cl.sub.2                                                     10    MeOH wash-40 ml. (2 times)                                                                            3                                                 11    Triethylamine(TEA) 12.5 percent in                                                                    3                                                       CH.sub.2 Cl.sub.2 -70 ml. (1 time)                                       ______________________________________                                    

After step 3, an aliquot may be taken for a ninhydrin test as well known in the art: if the test is negative, proceed to step 4 for removal of BOC-group prior to coupling of the next amino acid; if the test is positive or slightly positive, repeat steps 9 through 11.

The above schedule is used for coupling of each of the amino acids of the peptide of the invention after the first amino acid has been attached. N.sup.α Boc protection is used for each of the remaining amino acids throughout the synthesis. N.sup.α Boc-β-D-2NAL is prepared by a method known in the art, e.g. as described in detail in U.S. Pat. No. 4,234,571, issued Nov. 18, 1980 or commercially available from SyntheTech, Oregon, U.S.A. The side chains of Lys in the 5-position and of D-Lys in the 6-position are protected with Fmoc. Bzl (benzyl ether) is used as a side chain protecting group for the hydroxyl group of Ser. Boc-Lys(Ipr) is used for the 8-position. After deblocking the α-amino group at the N-terminal using trifluoroacetic acid (TFA), acetylation is achieved using a large excess of acetic anhydride in dichloromethane.

Following completion of the assembly of the peptide and acetylation of the N-terminus, the following intermediate is present: Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Lys(Fmoc)-D-Lys(Fmoc)-Leu-Lys(Ipr)-Pro-D-Ala-NH-[MBHA resin support]. The unnatural amino acids in the 5- and 6-positions are formed by simultaneously carrying out the following reactions with the deprotected side chains of the Lys residues. The Fmoc protecting group is removed from both by treatment of the peptidoresin with 20 percent piperidine in DMF for 5 minutes, then washing with DMF, then treatment with more piperidine/DMF for 20 minutes. After washing the resin with DMF, CH₃ OH and finally DMF, the newly freed amino group is treated with a large excess (>10 fold) of diphenyl cyanocarbonimidate (PCI) in DMF. Thereafter, the peptide is then subjected to the standard wash (see Steps 10-11) and then treated with isopropylamine dissolved in DMF for 24 hours at about 22° C. to complete the formation of the aminoisopropyl cyanoguanidino moiety; for some of the more hindered reactants, this step may be repeated.

The cleavage of the peptide from the resin and deprotection of the Ser side chain takes place very readily at 0° C. with HF. Anisole is added as a scavenger prior to HF treatment. After the removal of HF under vacuum, the resin is extracted with 50% acetic acid, and the washings are lyophilized to provide a crude peptide powder.

Purification of the peptide is then effected by high performance liquid chromatography (HPLC), as known in the art and specifically set forth in J. Rivier, et al. J. Chromatography, 288 (1984) 303-328.

The peptide is judged to be homogeneous using thin layer chromatography and several different solvent systems, as well as by using reversed-phase high pressure liquid chromatography and an aqueous triethylammonium phosphate solution plus acetonitrile. Amino acid analysis of the resultant, purified peptide is consistent with the formula for the prepared structure, showing substantially integer-values for each amino acid in the chain; mass spectral analysis is also consistent. The optical rotation is measured on a photoelectric polarimeter as

    [α].sub.D.sup.20 =-2.8±0.5(c═1, 50% acetic acid).

The other peptides in Table I are similarly synthesized and purified. The peptides are assayed in vivo and may also be tested in vitro. If performed, in vitro testing is carried out using dissociated rat pituitary cells maintained in culture for 4 days prior to the assay. The levels of LH mediated in response to the application of peptides is assayed by specific radioimmunoassay for rat LH. Control dishes of cells only receive a measure which is 3 nanomolar in GnRH; experimental dishes receive a measure 3 nanomolar in GnRH plus a measure having either the present standard antagonist for comparison purposes i e [Ac-dehydro Pro¹, (4F)D-Phe², D-Trp³,6 ]-GnRH or the test peptide, in concentrations ranging from 0.01 to 10 nanomolar. The amount of LH secreted in the samples treated only with GnRH is compared with that secreted by the samples treated with the peptide plus GnRH. The ability of the test peptide to reduce the amount of LH released by 3 nanomolar GnRH is compared to that of the present standard peptide.

The in vivo testing determines effectiveness to prevent ovulation in female rats. In this test, a specified number of mature female Sprague-Dawley rats, e.g. five to ten, each having a body weight from 225 to 250 grams, is injected with a specified microgram dosage of peptide in either saline, bacteriostatic water, polyethylene glycol, corn oil or mixtures of the above with ethanol at about noon on the day of proestrus. Proestrus is the afternoon of ovulation. A separate female rat group is used as a control to which the peptide is not administered. Each of the control female rats ovulates on the evening of proestrus; of the rats treated, the number of them which ovulate is recorded. Each of the peptides is considered to be totally effective to prevent ovulation of female rats at a dose of about 500 micrograms.

All peptides listed in Table I are considered effective to block GnRH-induced LH secretion in vitro at some reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages. The following Table A shows the results of in vivo testing of various of these GnRH antagonists with the dosages being given in micrograms:

                  TABLE A                                                          ______________________________________                                         Peptide          Rats              Rats                                        No.      Dosage  Ovulating   Dosage                                                                               Ovulating                                   ______________________________________                                         1.       2.5     0/9         1.0   0/5                                         2.       2.5     6/6         2.0   2/10                                        3.       2.5     7/9         2.0   1/10                                        4.       2.5     0/6         2.0   1/10                                        5.       2.0      1/10                                                         6.       2.5     0/6         1.0   7/17                                        7.       2.5      3/20                                                         8.       2.5      5/19       1.0   5/5                                         9.       2.5     0/6         1.0   5/5                                         10.      2.5     5/8         2.0   1/10                                        12.      2.5     6/7         5.0   9/10                                        13.      5.0      5/12       10.0  3/5                                         15.      2.5     0/4         1.0   8/18                                        ______________________________________                                    

EXAMPLE II

Peptides as indicated in TABLE II having the formula: Ac-dehydroPro-(A)D-Phe-AA₃ -Ser-AA₅ -β-D-2NAL-Leu-AA₈ -Pro-D-Ala-NH₂ are prepared by the solid-phase procedure referred to above.

                  TABLE II                                                         ______________________________________                                         A           AA.sub.3    AA.sub.5  AA.sub.8                                     ______________________________________                                         17   4Cl        β-D-2NAL                                                                              Tyr     Lys(bcg)                                   18   "          "           "       Lys(ecg)                                   19   4F         (1For)D-Trp (2F)Phe Orn(ecg)                                   20   "          "           Tyr     Dpr(2ncg)                                  21   "          "           (2NO.sub.2)Phe                                                                         Dbu(icg)                                   22   "          (1Ac)D-Trp  (2CH.sub.3)Phe                                                                         Dbu(2mpcg)                                 23   4Br        "           Tyr     Dbu(3mpcg)                                 24   "          "           (2Br)Phe                                                                               Dbu(4mpcg)                                 25   H          D-Trp       (2Cl)Phe                                                                               Orn(2mncg)                                 26   4NO.sub.2  (5CH.sub.3)D-Trp                                                                           (3CH.sub.3)Phe                                                                         Orn(hcg)                                   27   "          (5F)D-Trp   His     Lys(tcg)                                   28   2,4Cl.sub.2                                                                               (5Cl)D-Trp  (3F)Phe Dpr(trcg)                                  29   "          (6NO.sub.2)D-Trp                                                                           (3Br)Phe                                                                               Orn(1ncg)                                  30   C.sup.α Me/4Cl                                                                      (5OCH.sub.3)D-Trp                                                                          (3I)Tyr Orn(pcg)                                   31   3,4Cl.sub.2                                                                               (5NH.sub.2)D-Trp                                                                           (3Cl)Phe                                                                               Dpr(chcg)                                  ______________________________________                                    

All peptides listed in Table II are considered effective to block GnRH-induced LH secretion in vitro at some reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE III

Peptides as indicated in TABLE III having the formula: G-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-Tyr-AA₆ -Leu-AA₈ -Pro-AA₁₀ are prepared by the solid-phase procedure referred to above.

                  TABLE III                                                        ______________________________________                                         G          AA.sub.6   AA.sub.8   AA.sub.10                                     ______________________________________                                         32     Ac      D-Arg      Lys(bcg) D-Ala--NH.sub.2                             32A    Ac      D-Arg      Lys(tcg) D-Ala--NH.sub.2                             33     Ac      D-Lys(bcg) Arg      "                                           34     For     D-Tyr      Lys(icg) Gly--NH.sub.2                               35     Bz      (Et)D-Arg  Orn(icg) "                                           36     Ac      D-Lys      Orn(ecg) "                                           37     Vac     D-Har      Orn(mcg) "                                           38     Acr     (4gua)D-Phe                                                                               Dpr(2ncg)                                                                               AzaGly--NH.sub.2                            39     Ac      D-Orn      Dpr(chcg)                                                                               D-Ala--NH.sub.2                             40     Acr     D-His      Dpr(tcg) "                                           41     Ac      (Bu)D-Arg  Dbu(1mncg)                                                                              "                                           42     "       (Bz)D-Orn  Dbu(2mpcg)                                                                              "                                           43     Vac     (4NH.sub.2)D-Phe                                                                          Dbu(4mpcg)                                                                              "                                           44     Bz      (Ac)D-Lys  Dbu(trcg)                                                                               AzaGly--NH.sub.2                            ______________________________________                                    

All peptides listed in Table III are considered effective to block GnRH-induced LH secretion in vitro at some reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE IV

Peptides as indicated in TABLE IV having the formula: Ac-AA₁ -(4Cl)D-Phe-D-3PAL-Ser-Tyr-D-Arg-AA₇ -AA₈ -Pro-D-Ala-NH₂ are prepared by the solid-phase procedure referred to above.

                  TABLE IV                                                         ______________________________________                                         AA.sub.1         AA.sub.7   AA.sub.8                                           ______________________________________                                         45     β-D-2NAL Leu        Lys(bcg)                                       46     (1Ac)D-Trp    Met        Lys(2mpcg)                                     47     (6Br)D-Trp    Tyr        Lys(3mpcg)                                     48     (5F)D-Trp     Nle        Lys(4mpcg)                                     49     (6NO.sub.2)D-Trp                                                                             Met        Orn(chcg)                                      50     (5Cl)D-Trp    Tyr        Orn(pcg)                                       51     (4Cl)D-Phe    Phe        Dpr(pcg)                                       52     (4F)D-Phe     (4F)Phe    Dpr(tcg)                                       53     (2,4Cl.sub.2)D-Phe                                                                           NML        Dbu(mcg)                                       54     dehydroPro    Nle        Dbu(2ncg)                                      55     β-D-2NAL Leu        Lys(Ocg)                                       56     (6OCH.sub.3)D-Trp                                                                            (1For)Trp  Orn(1ncg)                                      57     (5NH.sub.2)D-Trp                                                                             Nva        Orn(ecg)                                       58     (4NO.sub.2)D-Phe                                                                             NML        Orn(1mncg)                                     59     dehydroPro    (6NO.sub.2)Trp                                                                            Dbu(hicg)                                      ______________________________________                                    

All peptides listed in Table IV are considered effective to block GnRH-induced LH secretion in vitro at some reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE V

Peptides as indicated in TABLE V having the formula: Ac-AA₁ -(4Cl)D-Phe-AA₃ -Ser-AA₅ -AA₆ -Leu-ILys-Pro-D-Ala-NH₂ are prepared by the solid-phase procedure referred to above.

                  TABLE V                                                          ______________________________________                                         AA.sub.1  AA.sub.3   AA.sub.5   AA.sub.6                                       ______________________________________                                         60  β-D-2NAL                                                                            D-3PAL     Lys(bcg) β-D-2NAL                                61  "         (6NO.sub.2)D-Trp                                                                          "        (Dnp)D-His                                   62  "         D-Trp      Lys(ecg) (4gua)D-Phe                                  63  dehydroPro                                                                               β-D-2NAL                                                                             Orn(ecg) (6NO.sub.2)D-Trp                             64  "         β-D-1NAL                                                                             Orn(mcg) D-Val                                        65  β-D-2NAL                                                                            (1For)D-Trp                                                                               Orn(tcg) (Pr)D-Arg                                    66  "         "          Dbu(bcg) (5NH.sub.2)D-Trp                             67  dehydroPro                                                                               D-Trp      Dbu(2mpcg)                                                                              D-Tyr                                        68  "         D-2PAL     Dbu(4mpcg)                                                                              D-Nle                                        69  "         (1Ac)D-Trp Dbu(hcg) (4F)D-Phe                                    70  Pro       D-3PAL     Lys(pcg) β-D-1NAL                                71  (1For)D-  "          Lys(chcg)                                                                               (4NHCH.sub.3)D-Phe                               Trp                                                                        72  β-D-2NAL                                                                            "          Dpr(hcg) (Ac)D-Orn                                    73  "         "          Dpr(Ocg) (4NH.sub.2)D-Phe                             74  β-D-1NAL                                                                            (6Br)D-Trp Dpr(tcg) (1For)D-Trp                                  75  (6CH.sub.3)D-                                                                            D-4PAL     Dpr(bzcg)                                                                               D-4PAL                                           Trp                                                                        ______________________________________                                    

The peptides listed in Table V are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE VI

Peptides as indicated in TABLE VI having the formula: G-AA₁ -(4Cl)D-Phe-D-Trp-Ser-Tyr-AA₆ -Leu-AA₈ -Pro-D-Ala-NH₂ are prepared by the solid-phase procedure referred to above.

                  TABLE VI                                                         ______________________________________                                         G         AA.sub.1   AA.sub.6     AA.sub.8                                     ______________________________________                                         76   Ac       dehydroPro D-Lys(bcg) Lys(bcg)                                   77   Ac       β-D-2NAL                                                                             β-D-2NAL                                                                             Lys(3mpcg)                                 78   Ac       β-D-2NAL                                                                             D-Val      Lys(tcg)                                   79   Acr      Pro        D-Ser(OtBu)                                                                               Lys(chcg)                                  80   H        dehydroPro (imBzl)D-His                                                                              Lys(ecg)                                   81   Bz       (4Br)D-Phe (5Cl)D-Trp Orn(ecg)                                   82   "        D-pGlu     (6Br)D-Trp Orn(bzcg)                                  83   For      β-D-1NAL                                                                             (Me)D-Arg  Orn(Ocg)                                   84   "        dehydroPro D-Har      Orn(4mpcg)                                 85   Vac      β-D-2NAL                                                                             (Bz)D-Lys  Orn(chcg)                                  86   Ac       β-D-(Arg.sup.5)                                                                      β-D-2NAL                                                                             Lys(Ocg)                                   87   H        dehydroPro D-Ala      Lys(tcg)                                   ______________________________________                                    

The peptides listed in Table VI are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE VII

Peptides as indicated in TABLE VII having the formula: Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA₅ -AA₆ -Leu-AA₈ -Pro-D-Ala-NH₂ are prepared by the solid-phase procedure referred to above.

                  TABLE VII                                                        ______________________________________                                         AA.sub.5        AA.sub.6     AA.sub.8                                          ______________________________________                                          88     Arg         D-Lys(bcg)   Arg                                            89     "           D-Lys(Ocg)   "                                              90     Orn(bcg)    (4gua)D-Phe  (Et.sub.2)Arg                                  91     Lys(2ncg)   D-Lys(2ncg)  ILys                                           92     Orn(bzcg)   D-Lys(bzcg)  Har                                            93     Lys(act)    D-Lys(act)   ILys                                           94     Lys(hicg)   D-Lys(hicg)  ILys                                           95     Lys(trcg)   D-Lys(trcg)  ILys                                           96     Lys(bcg)    D-Lys(bcg)   ILys                                           97     "           D-3PAL       (EtPr)Har                                      98     "           D-Lys(Nic)   (Me.sub.2)Arg                                  99     Orn(tcg)    D-Lys(tcg)   (MeBu)Arg                                     100     Lys(Sbcg)   D-Lys(Sbcg)  ILys                                          ______________________________________                                    

The peptides listed in Table VII are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE VIII

Peptides as indicated in TABLE VIII having the formula: Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA₅ -AA₆ -Leu-AA₈ -Pro-D-Ala-NH₂ are prepared by the solid-phase procedure referred to above.

                  TABLE VIII                                                       ______________________________________                                         AA.sub.5         AA.sub.6     AA.sub.8                                         ______________________________________                                         101     Orn(bcg)     D-Orn(bcg)   ILys                                         101A    Lys(bcg)     D-Lys(bcg)   ILys                                         102     Orn(2mpcg)   D-Orn(2mpcg) ILys                                         103     Orn(tcg)     D-Orn(tcg)   ILys                                         103A    Aph(tcg)     D-Aph(tcg)   ILys                                         103B    Lys(tcg)     D-Lys(tcg)   ILys                                         104     Orn(bcg)     β-D-2NAL                                                                               ILys                                         104A    Aph(bcg)     D-Aph(bcg)   ILys                                         105     Orn(2mpcg)   β-D-2NAL                                                                               ILys                                         105A    Lys(Nic)     D-Lys(Nic)   Lys(tcg)                                     106     Lys(Nic)     D-Lys(Nic)   Lys(bur)                                     106A    Lys(tcg)     D-Lys(Nic)   ILys                                         106B    Lys(Nic)     D-Lys(tcg)   ILys                                         107     Lys(2ncg)    D-Lys(2ncg)  ILys                                         108     Lys(bzcg)    D-Lys(bzcg)  ILys                                         109     Lys(icg)     D-Lys(icg)   Arg                                          110     Orn(icg)     D-Orn(icg)   ILys                                         ______________________________________                                    

As a further working example, a solid phase synthesis is set forth hereinafter of peptide No. 103A above, which is referred to as [Ac-β-D-2NAL¹, (4Cl)D-Phe², D-3PAL³, Aph(tcg)⁵, D-Aph(tcg)⁶, ILys⁸, D-Ala¹⁰ ]-GnRH. This peptide has the following formula:

Ac-β-D-2NAL-(4Cl)D-phe-D-3PAL-Ser-Aph(3-amino 1,2,4 triazole)-D-Aph (3-amino 1,2,4 triazole)-Leu-Lys (isopropyl)-Pro-D-Ala-NH₂.

An MBHA resin is used, and Boc-protected D-Ala is coupled to the resin over a 2-hour period in CH₂ Cl₂ using a 3-fold excess of Boc derivative and DCC as an activating reagent. The D-Ala residue attaches to the MBHA resin by an amide bond.

Following the coupling of each amino acid residue, washing, deblocking and coupling of the next amino acid residue is carried out in accordance with the schedule set forth hereinbefore, using an automated machine and beginning with about 5 grams of resin. The side chain primary amino groups of Aph in the 5-position and of D-Aph in the 6-position are protected by Fmoc. Bzl(benzyl ether) is used as a side chain protecting group for the hydroxyl group of Ser. Boc-Lys(Ipr,Z) is used for the 8-position. After deblocking the α-amino group at the N-terminus using trifluoroacetic acid (TFA), acetylation is achieved using a large excess of acetic anhydride in dichloromethane.

Following completion of the assembly of the peptide and acetylation of the N-terminus, the following intermediate is present: Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Aph(Fmoc)-D-Aph(Fmoc)-Leu-Lys(Ipr,Z)-Pro-D-Ala-NH-[MBHA resin support]. The unnatural amino acids in the 5- and 6-positions are formed by simultaneously carrying out the following reactions with the deprotected side chains of the Aph residues. The Fmoc protecting group is removed from both by treatment of the peptidoresin with 20 percent piperidine in DMF for 5 minutes, then washing with DMF, then treatment with more piperidine/DMF for 20 minutes. After washing the resin with DMF, CH₃ OH and finally DMF, the newly freed amino group is treated with a large excess (>10 fold) of diphenyl cyanocarbonimidate(PCI) in DMF. Thereafter, the peptide is then subjected to the standard wash and then treated with hydrazine, dissolved in DMF, for 24 hours at about 22° C. to complete the formation of the cyanoguanidino moiety; this step is preferably repeated. The cyanoguanidino moieties that are formed spontaneously convert to the corresponding heterocycle, i.e. 3-amino, 1,2,4 triazole. The formation of the triazole moieties can take place anytime after the peptide chain has been elongated to include the 5-position residue, and there may be an advantage in forming these moieties prior to adding D-3PAL to avoid the possibility of potential side reactions.

The cleavage of the peptide from the resin and deprotection of the Ser and the Lys side chains takes place very readily at 0° C. with HF. Anisole is added as a scavenger prior to HF treatment. After the removal of HF under vacuum, the resin is extracted with 5% acetic acid, and the washings are lyophilized to provide a crude peptide powder.

Purification of the peptide is then effected by high performance liquid chromatography (HPLC), as known in the art and specifically set forth in J. Rivier, et al. J. Chromatography, 288 (1984) 303-328.

The peptide is judged to be homogeneous using capillary zone electrophoresis (CZE), as well as by using reversed-phase high performance liquid chromatography and an aqueous triethylammonium phosphate solution plus acetonitrile. Amino acid analysis of the resultant, purified peptide is consistent with the formula for the prepared structure, showing substantially integer-values for each amino acid in the chain; mass spectral analysis is also consistent. The optical rotation is measured on a photoelectric polarimeter as

    [α].sub.D.sup.20 =-33±1.0(c=1, 50% acetic acid).

In addition to the above synthesis, Peptide 103A is also synthesized with glycinamide at the C-terminus instead of D-Ala-NH₂ ; biological testing in vivo shows it is slightly less biologically potent than Peptide 103A.

Peptide 103A was also synthesized with the N-ethylamide at the C-terminus instead of D-Ala-NH₂ ; biological testing in vivo showed it exhibited just slightly less biological potency than that reported in TABLE B for Peptide 103B. In addition, Peptide 104A is also synthesized with the glycinamide at the C-terminus instead of D-Ala-NH₂ ; biological testing in vivo shows somewhat less biological potency than that reported in TABLE B for Peptide 104A.

All peptides listed in Table VIII are considered effective to block GnRH-induced LH secretion in vitro at some reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

In addition to the in vivo testing, mentioned above, to determine effectiveness to prevent ovulation in female rats, groups of about 6 adult castrate male Sprague-Dawley rats, each having a body weight from 225 to 250 grams, are each injected IV with a 50 microgram dosage of either Peptide No. 103A or a standard antagonist referred to as the Nal-Glu antagonist (see J. Clin. Edno. Metab., 71, 4, 881-888 (1990)) in either corn oil or a phosphate buffer in the presence of BSA (bovine serum albumin). A separate rat group is used as a control, to which only the carrier is administered. Each of the control rats and the rats being treated are monitored for LH levels in the bloodstream at about 3 hours, 24 hours, 36 hours and 48 hours following this single bolus injection. Peptide No. 103A is surprisingly effective to suppress the level of circulating LH in the bloodstream of male rats, at levels very substantially below that of the control and, after 12 hours, below that of the rats treated with the Nal-Glu antagonist. This very long-acting duration of biological effect was truly surprising.

EXAMPLE IX

Peptides as indicated in TABLE IX having the formula: Ac-β-D-2NAL-(4Cl)D-Phe-AA₃ -Ser-AA₅ -AA₆ -Leu-AA₈ -Pro-D-Ala-NH₂ are prepared by the solid-phase procedure referred to above.

                  TABLE IX                                                         ______________________________________                                         AA.sub.3       AA.sub.5  AA.sub.6   AA.sub.8                                   ______________________________________                                         111   D-Lys(bcg)   Tyr       D-Arg    Arg                                      111A  D-Aph(icg)   Tyr       D-3PAL   ILys                                     112   D-Lys(2mpcg) Tyr       D-Arg    Arg                                      113   D-Lys(tcg)   Tyr       D-Arg    Arg                                      113A  D-Lys(tcg)   Tyr       D-Lys(tcg)                                                                              ILys                                     114   D-Lys(bcg)   Arg       β-D-2NAL                                                                           Arg                                      114A  D-Lys(2mpcg) Arg       β-D-2NAL                                                                           Arg                                      115   D-Lys(tcg)   Arg       β-D-2NAL                                                                           Arg                                      116   D-Lys(bcg)   Tyr       D-Arg    ILys                                     117   D-Lys(tcg)   Tyr       D-Arg    ILys                                     117A  D-Lys(tcg)   Tyr       D-3PAL   Arg                                      117B  D-Aph(tcg)   Tyr       D-3PAL   ILys                                     118   D-Lys(bcg)   Tyr       β-D-2NAL                                                                           ILys                                     119   D-Lys(bcg)   Tyr       D-3PAL   ILys                                     119A  D-Aph(bcg)   Tyr       D-3PAL   ILys                                     120   D-Lys(2mpcg) Tyr       D-3PAL   ILys                                     120A  D-Lys(2mpcg) Arg       β-D-2NAL                                                                           Arg                                      121   D-Lys(bcg)   Tyr       D-Arg    Arg                                      122   D-Lys(bcg)   Lys(bcg)  D-Lys(bcg)                                                                              ILys                                     122A  D-Lys(bcg)   Tyr       D-Lys(bcg)                                                                              ILys                                     123   D-3PAL       Lys(bur)  β-D-2NAL                                                                           Arg                                      124   D-Lys(bcg)   Tyr       β-D-2NAL                                                                           Lys(bcg)                                 125   D-Orn(2mpcg) Tyr       β-D-2NAL                                                                           ILys                                     125A  D-Orn(tcg)   Tyr       D-3PAL   ILys                                     125B  D-Orn(bcg)   Tyr       D-3PAL   ILys                                     ______________________________________                                    

All peptides listed in Table IX are considered effective to block GnRH-induced LH secretion in vitro at some reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE X

Peptides as indicated in TABLE X having the formula: Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-AA₅ -AA₆ -AA₇ -ILy-Pro-AA₁₀ are prepared by the solid-phase procedure referred to above.

                  TABLE X                                                          ______________________________________                                         AA.sub.5     AA.sub.6    AA.sub.7 AA.sub.10                                    ______________________________________                                         125C  Aph(tcg)   D-Aph(tcg)  NML    D-Ala--NH.sub.2                            125D  Aph(tcg)   D-Aph(tcg)  Leu    NHCH.sub.2 CH.sub.3                        125E  Aph(tcg)   D-Aph(tcg)  NML    "                                          125F  Aph(tcg)   D-Aph(tcg)  Leu    Gly--NH.sub.2                              125G  Aph(2mpcg) D-Aph(2mpcg)                                                                               NML    D-Ala--NH.sub.2                            ______________________________________                                    

The peptides listed in Table X are considered effective to block GnRH-induced LH secretion in vitro at a reasonable concentration. All of the peptides are considered to be effective to prevent ovulation of female mammals at low dosages.

Results of in vivo testing of selected of these antagonists are shown in the following Table B, with the dosages being given in micrograms:

                  TABLE B                                                          ______________________________________                                         Peptide          Rats              Rats                                        No.      Dosage  Ovulating   Dosage                                                                               Ovulating                                   ______________________________________                                          17.     10      6/8         15     7/10                                        32A.    10      0/6         5     0/6                                                  2.5     0/7         1.0    5/10                                        33.     2.5      4/17                                                          55.     10      6/7         25    2/5                                          60.     2.5      4/14       1.0    8/10                                        86.     2.5      4/15       5.0   5/5                                          88.     2.5     0/4         1.0    4/15                                                10      0/8                                                            89.     10       2/18                                                          93.     2.5      5/10                                                          94.     1.0      7/11                                                          96.     1.0      9/12                                                         101.     2.5     0/5         1.0   5/8                                         102.     2.5     2/9                                                           103.     2.5     5/7                                                           103A.    2.5     0/8         1.0   0/7                                                                      0.5   7/8                                         103B.    2.5     0/7         1.0   3/9                                                                      0.5   7/8                                         104.     2.5     6/8                                                           104A.    5.0      6/11       2.5   2/3                                         105.     2.5     0/5                                                           105A.    5.0     4/6                                                           106.     5.0     0/7                                                           106A.    2.0     0/5         1.0   7/7                                         106B.    2.5     0/6         1.0   4/6                                         111.     2.5      1/11       1.0   0/5                                                                      0.5   5/7                                         111A.    15      0/9         5     2/4                                         112.     1.0     4/8                                                           113.     1.0     5/6                                                           113A.    5.0     0/6         2.5    1/15                                       114.     5.0     4/5                                                           115.     2.5     2/8                                                           116.     2.5     0/6         1.0   2/8                                         117.     10      0/6         5     2/5                                         117A.    2.5     0/7         1.0   4/9                                         117B.    15      1/5                                                           118.     15      4/5         5     5/5                                         119.     2.5     0/7         1.0    2/10                                       119A.    15      4/6                                                           120.     2.5     0/8         1.0   6/7                                         120A.    5.0     4/6                                                           122.     10      4/6                                                           122A.    5.0     0/6         2.5   5/6                                         123.     10       1/10       5     0/6                                         125.     2.5     3/7                                                           125A.    5.0     1/8         2.5   1/7                                         125B.    2.5     0/7                                                           125C.    1.0     0/8         0.5    6/10                                       125D.    2.5     0/8         1.0   8/9                                         125E.    10      0/5                                                           125F.    1.0      9/16                                                         125G.    5.0     0/6                                                           ______________________________________                                    

EXAMPLE XI

Peptides as indicated in TABLE XI having the formula: pGlu-His-Trp-Ser-Tyr-AA₆ -AA₇ -Arg-Pro-AA₁₀ are prepared by the solid phase procedure referred to above.

                  TABLE XI                                                         ______________________________________                                         AA.sub.6         AA.sub.7  AA.sub.10                                           ______________________________________                                         126     D-Lys(bcg)   Leu       Gly--NH.sub.2                                   126A    D-Aph(bcg)   "         "                                               127     D-Lys(ecg)   "         "                                               128     D-Lys(pcg)   "         "                                               129     D-Lys(hcg)   "         AzaGly--NH.sub.2                                130     D-Lys(tcg)   "         NHCH.sub.2 CH.sub.3                             130A    D-Aph(tcg)   "         "                                               131     D-Lys(4mpcg) "         "                                               132     D-Lys(3mpcg) NML       "                                               133     D-Lys(2mpcg) "         "                                               134     D-Lys(chcg)  Leu       Gly--NH.sub.2                                   134A    D-Aph(tcg)   "         "                                               135     D-Lys(Ocg)   "         "                                               136     D-Orn(bcg)   "         "                                               137     D-Orn(tcg)   NML       AzaGlyNH.sub.2                                  138     D-Orn(mcg)   "         NHCH.sub.2 CH.sub.3                             139     D-Dbu(2mpcg) "         NHCH.sub.3                                      140     D-Dbu(chcg)  Leu       NHCH.sub.2 CH.sub.2 CH.sub.3                    141     D-Dbu(bzcg)  "         Gly--NH.sub.2                                   142     D-Dpr(ecg)   "         "                                               143     D-Dpr(hicg)  NML       "                                               144     D-Dpr(trcg)  "         NHCH.sub.2 CH.sub.3                             ______________________________________                                    

The peptides described in TABLE XI are considered to be effective to cause the release of LH and FSH in female rats. All of them are considered to be substantial more effective than native GnRH.

An exemplary synthesis of Peptide No. 130A which has the formula pGlu-His-Trp-Ser-Tyr-D-Aph(3-amino-1,2,4-triazole)-Leu-Arg-Pro-NHCH₂ CH₃ is set forth hereinafter.

An N-ethylamino methyl resin is used, and Boc-protected Pro is coupled to the resin over a 2-hour period in CH₂ Cl₂ using a 3-fold excess of Boc derivative and DCC as an activating reagent. The Pro residue attaches by an amide bond to the secondary amino group on the resin.

Following the coupling of each amino acid residue, washing, deblocking and coupling of the next amino acid residue is carried out in accordance with the schedule set forth hereinbefore, using an automated machine and beginning with about 5 grams of resin. The side chain primary amino group of D-Aph in the 6-position is protected by Fmoc. Bzl(benzyl ether) is used as a side chain protecting group for the hydroxyl group of Ser, and 2,6-dichlorobenzyl is used as the side chain protecting group for the hydroxyl group of Tyr. Boc-Arg(Tos) is used for the 8-position, and Boc-His(Tos) is used for the 2-position. pGlu at the N-terminus is introduced as Z-pGlu or as plain pGlu, as desired.

Following completion of the assembly of the peptide, the following intermediate is present: pGlu-His(Tos)-Trp-Ser(Bzl)-Tyr(DCB)-D-Aph(Fmoc)-Leu-Arg(Tos)-Pro-N(Et)-[resin support]. The unnatural amino acid in the 6-position is formed by carrying out the following reactions with the deprotected side chain of the D-Aph residue. The Fmoc protecting group is removed by treatment of the peptidoresin with 20 percent piperidine in DMF for 5 minutes, then washing with DMF, then treatment with more piperidine/DMF for 20 minutes. After washing the resin with DMF, CH₃ OH, CH₂ Cl₂, and finally DMF, the newly freed amino group is treated with a large excess (>10 fold) of diphenyl cyanocarbonimidate(PCI) in DMF. Thereafter, the peptide is then subjected to the standard wash and then treated with hydrazine, dissolved in DMF, for 24 hours at about 22° C. to complete the formation of the cyanoguanidino moiety; this step is preferably repeated. The cyanoguanidino moieties that are formed spontaneously convert to the corresponding heterocycle, i.e. 3-amino, 1,2,4 triazole.

The cleavage of the peptide from the resin and deprotection of the pGlu, His, Ser, Tyr and Arg side chains takes place very readily at 0° C. with HF. Anisole is added as a scavenger prior to HF treatment. After the removal of HF under vacuum, the resin is extracted with 50% acetic acid, and the washings are lyophilized to provide a crude peptide powder.

Purification of the peptide is then effected by high performance liquid chromatography (HPLC), as known in the art and specifically set forth in J. Rivier, et al. J. Chromatography, 288 (1984) 303-328.

The peptide is judged to be homogeneous using capillary zone electrophoresis (CZE), as well as by using reversed-phase high performance liquid chromatography and an aqueous triethylammonium phosphate solution plus acetonitrile. Amino acid analysis of the resultant, purified peptide is consistent with the formula for the prepared structure, showing substantially integer-values for each amino acid in the chain; mass spectral analysis is also consistent.

In addition to the above synthesis, Peptide 134A is also synthesized with glycinamide at the C-terminus instead of the ethylamide; biological testing in vivo shows it is just slightly less biologically potent than Peptide 130A.

EXAMPLE XII

A peptide intermediate having the formula: Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Lys(Fmoc)-D-3PAL-NML-Lys(Dts)-Pro-D-Ala-NH-[resin support] is prepared by the solid phase procedure generally referred to above. The intermediate is then treated with piperidine to remove the Fmoc protecting group and is thereafter reacted with PCI as hereinbefore described. Next the Dts protecting group is removed from the amino side chain of the Lys residue in the 8-position using a suitable thiol, such as β-mercaptoethanol or thiophenol(PhSH), in DMF, and the peptidoresin is given the standard wash. Thereafter, it is maintained at 22° C. for 10-60 minutes or until the ninhydrin test is negative to allow the reaction to proceed to completion, effecting cyclization of the side-chain primary amino group of Lys⁸ and the cyanoguanidino moiety which was earlier formed upon the Lys⁵ side chain. Deprotection and cleavage are then carried out as previously described. Following HPLC purification as previously described, the GnRH antagonist (Peptide No. 145) is tested. The peptide is considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE XIII

A peptide intermediate having the formula: Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Orn(Fmoc)-D-Trp-Leu-Lys(Nps)-Pro-D-Ala-NH-[resin support] is prepared by the solid phase procedure referred to above. The peptide intermediate is treated with a suitable thiol as described in Example 145 to form the cyanoguanidino moiety by reaction of PCI with the side chain amino group of the Lys residue in the 8-position, and it is then cyclized with the deprotected side chain amino group of Orn in the 5-position following removal of the Fmoc protecting group. Following cleavage and HPLC purification as previously described, the GnRH antagonist (Peptide No. 146) is tested. The peptide is considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE XIV

A peptide intermediate having the formula: Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Tyr(2BrZ)-D-Lys(Fmoc)-Leu-Lys(Ipr)-Pro-D-Ala-NH-[resin support] is prepared by the solid phase procedure referred to above. Following the removal of the Fmoc protection, the peptide intermediate is reacted as generally described in Example I using naphthyl isocyanate instead of PCI to form the napthylurea moiety with the side chain amino group of the D-Lys residue in the 6-position. Following cleavage and HPLC purification as previously described, the GnRH antagonist (Peptide No. 147) is tested. The peptide is considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE XV

A peptide intermediate having the formula: Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Tyr(2BrZ)-D-Lys(Fmoc)-Leu-Lys(Ipr)-Pro-D-Ala-NH-[resin support] is prepared by the solid phase procedure referred to above. Following the removal of the Fmoc protection, the peptide intermediate is reacted as generally described in Example I using naphthyl isothiocyanate instead of PCI to form the napthylthiourea moiety with the side chain amino group of the residue in the 6-position. Following cleavage and HPLC purification as previously described, the GnRH antagonist (Peptide No. 148) is tested. The peptide is considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE XVI

A peptide intermediate having the formula: Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser(Bzl)-Tyr(2BrZ)-D-Lys(Fmoc)-Leu-Lys(Ipr)-Pro-D-Ala-NH-[resin support] is prepared by the solid phase procedure referred to above. Following the removal of the Fmoc protection, the peptide intermediate is first reacted using 2-bromoethyl,2'(Boc-amino)ethyl ether dissolved in DMF for 1 hour or until the ninhydrin test is negative to link the carbon atom to the side-chain amino group by the removal of the halogen to form: Q--NH--(CH₂)₂ --O--(CH₂)₂ --NH(Boc.sub.). This compound is then reacted as generally described in Example I using PCI to form the cyanoguanidino moiety with the side chain secondary amino group of the residue in the 6-position. Next the Boc protection group is removed, and the primary amino group reacts with the --OPh group to give the compound: ##STR32## Following cleavage and HPLC purification as previously described, the GnRH antagonist (Peptide No. 149) is tested. The peptide is considered to be effective to prevent ovulation of female mammals at low dosages.

EXAMPLE XVII

(Boc)Dpr is reacted with PCI as in Example I to produce ##STR33## hydrazine dissolved in DMF for 1 to 2 days at room temperature, washed with DMF and then repeated to replace the --OPh group with concomminent formation of the heterocycle: ##STR34## which is particularly useful as a substitute for His in peptide syntheses.

Following purification of the peptides, various of them are further characterized by subjection to high performance liquid chromatography on C₁₈ silica (Vydac 0.46×25 cm) using a flow rate of 1.7 ml/min and a gradient of from 35% to 85% by volume of Buffer B over a time span of 50 minutes, with the remainder being Buffer A. Buffer A is a solution of 0.3% triethylamine (v/v) and 0.1% phosphoric acid in water at pH 7.0; Buffer B is 60% by volume acetonitrile in Buffer A. The following Table C shows when the specific peptides elute from the C₁₈ silica having a particle size of about 5μ and a pore size of 300 Å when subjected to a gradient of from 35% Buffer B to 85% Buffer B (by volume with the remainder being Buffer A) over 50 minutes at a flow rate of 1.7 ml. per minute, and thereafter at 85% isocratically for 10 min.:

                  TABLE C                                                          ______________________________________                                         Peptide No.   Time of Elution                                                  ______________________________________                                          1.                                                                             2.           26.56                                                             3.           47.6                                                              4.           20.32                                                             5.           35.6                                                              6.           31.04                                                             7.           28.96                                                             8.           27.74                                                             9.           54.24                                                            10.           30.86                                                            11.           56.9                                                             12.           46.78                                                            13.           33.4                                                             15.           41.1                                                             17.           49.6                                                             32.           40.56                                                            33.           35.84                                                            ______________________________________                                    

The peptides of the invention are often administered in the form of pharmaceutically acceptable, nontoxic salts, such as acid addition salts, or of metal complexes, e.g., with zinc, barium, calcium, magnesium, aluminum or the like (which are considered as addition salts for purposes of this application), or of combinations of the two. Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, nitrate, oxalate, fumarate, gluconate, tannate, maleate, acetate, citrate, benzoate, succinate, alginate, malate, ascorbate, tartrate and the like. For example, an aqueous solution of the peptide can be repeatedly treated with 1N acetic acid and then lyophilized to yield the acetic acid salt thereof. If the active ingredient is to be administered in tablet form, the tablet may contain a pharmaceutically-acceptable diluent which includes a binder, such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring may be used as part of the pharmaceutically-acceptable diluent, and intravenous administration in isotonic saline, phosphate buffer solutions or the like may be effected.

The pharmaceutical compositions will usually contain the peptide in conjunction with a conventional, pharmaceutically-acceptable carrier. Usually, the dosage will be from about 10 micrograms to about 2.5 milligrams of the peptide per kilogram of the body weight of the host when given intravenously; although oral dosages will be higher, it is anticipated that the nature of these compounds will permit effective oral administration. Overall, treatment of subjects with these peptides is generally carried out in the same manner as the clinical treatment using other antagonists or agonists of GnRH using a suitable carrier in which the peptide is soluble.

It may also be desirable to deliver the GnRH analog over prolonged periods of time, for example, for periods of one week to one year from a single administration, and slow release, depot or implant dosage forms may be utilized. For example, a dosage form may contain a pharmaceutically acceptable non-toxic salt of the compound which has a low degree of solubility in body fluids, for example, an acid addition salt with a polybasic acid; a salt with a polyvalent metal cation; or combination of the two salts. A relatively insoluble salt may also be formulated in a gel, for example, an aluminum stearate gel. A suitable, slow-release depot formulation for injection may also contain the GnRH analog or a salt thereof dispersed or encapsulated in a slow degrading, non-toxic or non-antigenic polymer such as a polylactic acid/polyglycolic acid polymer, for example, as described in U.S. Pat. No. 3,773,919. These compounds may also be formulated into silastic implants.

These peptides can be administered to mammals intravenously, subcutaneously, intramuscularly, orally, percutaneously, e.g. intranasally or intravaginally to achieve fertility inhibition and/or control and also in applications calling for reversible suppression of gonadal activity, such as for the management of precocious puberty or during radiation- or chemotherapy. They are also useful for treatment of steroid-dependent tumors. Effective dosages will vary with the form of administration and the particular species of mammal being treated. An example of one typical dosage form is a bacteriostatic water solution containing the peptide which solution is administered parenterally to provide a dose in the range of about 0.1 to 2.5 mg/kg of body weight per day. Oral administration of the peptide may be given in either solid form or liquid form.

Although the invention has been described with regard to its preferred embodiments, it should be understood that changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims which are appended hereto. For example, other substitutions known in the art which do not significantly detract from the effectiveness of the peptides may be employed in the peptides of the invention. D-2PAL and D-4PAL are considered to be equivalents of D-3PAL. Instead of the amino group of Aph being situated at the para position, it may alternatively be located in the meta or ortho position. Other equivalent acylating groups can be used instead of acetyl at the N-terminus. The 6-position substitutes set forth in Table VI are considered to be equivalents known in the prior art and can be included in the peptides of the invention. Substituted Phe, such as (4F) Phe, can be used instead of Phe in the 7-position. Both butyl Lys and diethyl Lys are considered to be equivalents of ILys; however, ILys is preferred when neither U* or Arg is in the 8-position. Har is considered the equivalent of Arg in this position. Other hydrophobic amino acid residues can also be employed in the 1-position, preferably in D-isomer form, and are considered equivalents of those specified. Moreover, the analogs can be administered in the form of their pharmaceutically or vetinarially acceptable, nontoxic salts, as indicated hereinbefore, which are considered equivalents.

Particular features of the invention are emphasized in the claims which follow. 

What is claimed is:
 1. A method for making a peptide which includes at least one residue U* of an unnatural amino acid having the formula: ##STR35## which peptide has the formula: G-AA-AA₂ -AA'-Ser-AA₅ -AA₆ -AA₇ -AA₈ -Pro-AA₁₀ wherein G is hydrogen or an acyl group having 7 or less carbon atoms; AA is AA₁ or pGlu; AA₁ is dehydroPro, D-pGlu, (A) D-Phe, (B)D-Trp, Pro, or β-D-NAL; A is H, Cl, F, NO₂, CH₃, OCH₃, C.sup.α Me/4Cl, Cl₂ or Br; B is H, NO₂, NH₂, OCH₃, F, Cl, Br, CH₃, N^(in) For or N^(in) Ac; AA₂ is His or (A) D-Phe; AA' is AA₃ or Trp; AA₃ is U*, D-PAL, β-D-NAL or (B)D-Trp; AA₅ is U*, Lys(cpd), Orn(cpd), Dbu(cpd), Dpr(cpd), Tyr,(C)Arg, (A)Phe, (3I)Tyr or His; C is H or lower alkyl; AA₆ is U*, β-D-NAL, (B) D-Trp, (A')D-Phe, (D)D-Orn, (D)D-Lys, (D)Dbu, (D)D-Dpr, D-Har, D-Tyr, (E)D-His, D-PAL, (C)D-Arg, D-Leu, D-Ile, D-Nle, D-Val, D-Ala, or D-Ser(OtBu); A' is A, NH₂, NHCH₃ or gua; D is G, cpd or an aryl group; E is H, imBzl or dinitrophenol; AA₇ is Nle, Leu, NML, (A) Phe, Met, Nva, Tyr, (B)Trp or PAL; AA₈ is U*, ILys, (C')Arg or (C')Har; C' is H or di-lower alkyl; AA₁₀ is D-Ala-NH₂, Gly-NH₂, NHNHCONH₂ or NH(R); R is lower alkyl; provided, however, that at least one of AA₃, AA₅, AA₆ and AA₈ is U*, which method comprises (a) forming an intermediate peptide having the formula:X¹ -AA-AA₂ (X⁵)-U₃ -Ser (X³)-U₅ -U₆ -AA₇ (X² or X⁷)-U₈ -Pro-X⁸ wherein: U₃ is either U' or AA' (X²); U₅ is either U' or AA₅ (X⁴ or X⁵); U₆ is either U' or AA₆ (X⁴ or X⁵ or X⁶); U₈ is either U' or AA₈ (X⁵ or X⁶); U' is Aph(X⁸); X¹ is hydrogen or an α-amino protecting group; X² is hydrogen or a protecting group for an indole nitrogen; X³ is a protecting group for a hydroxyl group of Ser or Thr; X⁴ is hydrogen or a protecting group for a phenol ic hydroxyl group of Tyr; X⁵ is either hydrogen or a protecting group for a guanidino or imidazole group; X⁶ is a protecting group for a primary amino group; X^(a) is a protecting group for a primary amino group that is base-labile, hydrazine-labile or thio-labile; X⁷ is hydrogen or a protecting group for Met; X⁸ is Gly-NH-resin support, D-Ala-NH-resin support, N(A)-resin support, an amide either of Gly or of D-Ala or a substituted amide attached directly to Pro; provided however that at least one of U₃, U₅, U₆ and U₈ is Aph(X^(a)) , with U₃ and U₆ always being a D-isomer; (b) removing at least one X^(a) to selectively deprotect a side chain primary amino group of at least one Aph residue of said intermediate peptide; (c) reacting said deprotected Aph primary amino group to build said residue into one having the formula U* by first treating with diphenyl cyanocarbonimidate and subsequently treating with hydrazine to create amino-triazole moieties; and (d) splitting off any remaining groups X¹ to X⁷ and/or cleaving from any resin support included in X⁸.
 2. A method in accordance with claim 1 wherein either U₅ is U' or U₆ is U' or both are U'.
 3. A method in accordance with claim 1 wherein either U₃ or U₈ is U'.
 4. A method in accordance with claim 1 wherein U₅ and U₈ are U'.
 5. A method according to claim 1 for making a GnRH antagonist peptide having the formula: Ac-AA₁ -(A)D-Phe-AA₃ -Ser-AA₅ -AA₆ -AA₇ -AA₈ -Pro-AA₁₀ wherein AA₁ is (A)D-Phe, (B) D-Trp or β-D-2NAL; A is H, 4Cl, 4F, 4NO₂, 4CH₃, 4OCH₃, C.sup.α Me/4Cl, 2,4Cl₂ or 4Br; B is H, 6NO₂, 6NH₂, 6OCH₃, 6F, 6Cl, 6Br, 6CH₃, N^(in) For or N^(in) Ac; AA₃ is U*, D-3PAL, β-D-2NAL or (B) D-Trp; AA₅ is U*, Lys(cpd) or Tyr; AA₆ is U*, β-D-2NAL, 4NH₂ D-Phe, (B) D-Trp, D-Lys (cpd) , D-3PAL or D-Arg; AA₇ is Nle, Leu, NML or Phe; AA₈ is U* ILys or Arg; provided, however, that at least one of AA₃, AA₅, AA₆ and AA₈ is U*, with AA₃ and AA₆ always being a D-isomer.
 6. A method for making a GnRH antagonist peptide according to claim 5 wherein both AA₅ and AA₆ are U*.
 7. A method for making a GnRH agonist peptide according to claim 1 wherein only AA₆ is U*.
 8. A method according to claim 1 for making a GnRH antagonist peptide having the formula: Ac-AA₁ -(A)D-Phe-AA₃ -Ser-AA₅ -AA₆ -AA₇ -AA₈ -Pro-AA₁₀ wherein AA₁ is (A)D-Phe or β-D-2NAL; A is H, 4Cl, 1,4F, 4NO₂, 4CH₃, 4OCH₃, C.sup.α Me/4Cl, 2,4Cl₂ or 4Br; AA₃ is D-3PAL, β-D-2NAL or (B) D-Trp; B is H, 6NO₂, 6NH₂, 6OCH₃, 6F, 6Cl, 6Br, 6CH₃, N^(in) For or N^(in) Ac; AA₅ is U*; AA₆ is U*, AA₇ is Nle, Leu, NML or Phe; and AA₈ is ILys or Arg; wherein Aph residues in AA₅ and AA₆ are simultaneously modified to create amino triazole moieties.
 9. A method for making a peptide in accordance with claim 8 wherein AA₁ is β-D-2NAL, (A) is 4Cl or 4F and AA₃ is D-3PAL.
 10. A method for making a peptide in accordance with claim 9 wherein AA₈ is ILys.
 11. A method for making a GnRH antagonist peptide which includes at least one residue U* of an unnatural amino acid having the formula: ##STR36## which peptide has the formula: G-β-D-NAL- (A) D-Phe-D-PAL-Ser-AA₅ -AA₆ -Leu-AA₈ -Pro-AA₁₀ wherein G is hydrogen or an acyl group having 7 or less carbon atoms; A is H, Cl, F, NO₂, CH₃, OCH₃, C.sup.α Me/4Cl, Cl₂ or Br; AA₅ is U*; AA₆ is U*; AA₈ is ILys or Arg; AA₁₀ is D-Ala-NH₂, Gly-NH₂ or NHCH₂ CH₃ ; with AA₆ being a D-isomer, which method comprises (a) forming an intermediate peptide having the formula: X¹ -β-D-NAL- (A) D-Phe-D-PAL-Ser (X³)-Aph (X^(a))-D-Aph(X⁸)-Leu-AA₈ (X⁵ or X⁶)-Pro-X⁸ wherein X¹ is hydrogen or an α-amino protecting group; X³ is a protecting group for a hydroxyl group of Ser; X⁵ is either hydrogen or a protecting group for a guanidino group; X⁶ is a protecting group for an amino group; X^(a) is a protecting group for a primary amino group that is base-labile, hydrazine-labile or thio-labile; X⁸ is Gly-NH-resin support, D-Ala-NH-resin support or N(A)-resin support, (b) removing X^(a) to deprotect side chain primary amino groups of said Aph residues of said intermediate peptide without removing other protecting groups; (c) reacting said deprotected side chain primary amino groups to simultaneously build each said Aph residue into one having the formula U* by first treating with diphenyl cyanocarbonimidate and subsequently treating with hydrazine to create amino-triazole moieties; and (d) splitting off any remaining groups X¹ to X⁶ and cleaving from any resin support included in X⁸.
 12. A method according to claim 11 for making a GnRH antagonist peptide having the formula: Ac-β-D-2NAL-(A)D-phe-D-3pAL-Ser-AA₅ -AA₆ -Leu-AA₈ -Pro-D-Ala-NH₂ wherein AA₁ is (A) D-Phe; A is 4Cl,4F or 4Br; AA₅ is Aph(tcg); AA₆ is D-Aph(tcg) and AA₈ is ILys or Arg.
 13. A method for making a peptide in accordance with claim 12 wherein (A) is 4Cl or 4F.
 14. A method for making a peptide in accordance with claim 13 wherein AA₈ is ILys.
 15. A method for making a peptide in accordance with claim 12 wherein the decapeptide intermediate is formed and the N-terminus is deprotected and acetylated prior to removing X^(a) as a part of step (b).
 16. A method for making a peptide in accordance with claim 12 wherein X^(a) is Fmoc and is removed with piperidine.
 17. A method for making a peptide in accordance with claim 12 wherein said reaction with hydrazine is carried out in dimethylformamide at about 22° C.
 18. A method for making a peptide in accordance with claim 11 having the formula:Ac-β-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-Aph(3-amino 1,2,4 triazole)-D-Aph(3-amino 1,2,4 triazole)-Leu-Lys(isopropyl)-Pro-D-Ala-NH₂. 